The invention is directed toward methodologies and apparatus for use in the preparation of devitalized, i.e. essentially lacking in reproductively viable cells and/or metabolically viable cells, while preferably retaining reproductively non-viable cells and/or metabolically non-viable cells and/or large molecular weight cytoplasmic proteins including actin, soft-tissue implants, in small quantities and commercializable quantities. Such soft-tissue implants include vascular graft substitutes. These implants can be derived from tissue engineered soft tissue devices, tissue products derived from animal or human donors that contain cells, and that contain or are devoid of valve structures useful in directing the flow of fluids through tubular vascular devices, and/or combinations of natural tissue products and tissue engineered soft-tissue products. The invention includes methodologies and apparatus for producing uniform, gently processed, devitalized multiple soft tissue implants, where processing time is significantly reduced and the number of implants produced per day is increased. The devitalized grafts produced are significantly improved in long-term durability and function when used in clinical applications.
Numerous types of vascular graft substitutes have been produced in the last four decades. These vascular graft substitutes have included large and small diameter vascular, blood carrying tubular structures, grafts containing valvular structures (vein substitutes, and heart valve substitutes) and lacking valvular structures (artery substitutes). The materials out of which these vascular grafts have been constructed have included man-made polymers, notably Dacron and Teflon in both knitted and woven configurations, and non-man-made polymers, notably tissue engineered blood vessels such as described in U.S. Pat. Nos: 4,539,716, 4,546,500; 4,835,102; and blood vessels derived from animal or human donors such as described in U.S. Pat. Nos. 4,776,853; 5,558,875; 5,855,617; 5,843,181; and 5,843,180.
The prior art processing methods are directed to decellularizing tissue grafts, i.e. removing all cellular elements leaving a tissue matrix free from cellular elements, and are prohibitively time consuming, easily requiring numerous days, for example anywhere from eight to twenty-one days total processing time. Such long processing times result in proteolytic degradation of the matrix structures of the processed tissues. Over the past few decades numerous efforts have been made to manage the large surgical use of vascular prostheses in the treatment of vascular dysfunctions/pathologies. While vascular prostheses are available for clinical use, they have met with limited success due to cellular and immunological complications, and the inability to remain patent and function. These problems are especially pronounced for small diameter prostheses, i.e. less than about 6 mm. Efforts have been directed at removing those aspects of allograft and xenograft vascular prostheses that contribute to immunological xe2x80x9crejectionxe2x80x9d and these efforts have focused primarily on development of various xe2x80x9cdecellularizationxe2x80x9d processes, which processes require unduly burdensome incubation times. In addition the prior art methods involve using volumes of processing solutions which do not lend themselves to the production of large numbers of vascular grafts, which ability to xe2x80x9cscale-upxe2x80x9d is necessary for economic clinical use.
The inventive process produces devitalized grafts including but not limited to ligaments, tendons, menisci, cartilage, skin, pericardium, dura mater, fascia, small and large intestine, placenta, veins, arteries, and heart valves. The process is advantageous over prior art processes in that processing times and conditions have been optimized and reduced, and the economics of production have been dramatically improved, resulting in large numbers of uniform, non-immunogenic grafts being produced. The grafts produced are non-immunogenic, are substantially free from damage to the matrix, and are substantially free from contamination including for example free from infectious agents.
The invention involves the use of one or more non-denaturing agent, for example N-lauroylsarcosinate, for the treatment of tissues with the dual objective of devitalization and treatment of tissues to enhance recellularization upon implantation.
The invention is directed at a process for producing devitalized soft-tissue implants including vascular grafts, veins, arteries, and heart valves, where processing times and conditions have been optimized to dramatically improve on the economics of production as well as to produce a graft with minimum damage to the matrix structure of the devitalized graft.
The inventive process is a process for preparing biological material(s) for implantation into a mammalian cardiovascular system, musculoskeletal system, or soft tissue system. The process removes reproductively viable cells and/or metabolically viable cells, while preferably retaining reproductively non-viable cells and/or metabolically non-viable cells and/or large molecular weight cytoplasmic proteins including for example, actin.
The process provides for the production of commercializable quantities of devitalized soft tissue grafts for implantation into mammalian systems by removing reproductively viable cells and/or metabolically viable cells, while preferably retaining reproductively non-viable cells and/or metabolically non-viable cells and/or large molecular weight cytoplasmic proteins, such proteins including actin, forming an devitalized non-soluble matrix, the matrix having as major components collagens, elastins, hyaluronins, and proteoglycans. The devitalized tissue produced can be implanted into a mammalian system and recellularized in vitro, or recellularized in vitro and subsequently implanted into a mammalian system.
An embodiment of the process includes the following steps:
isolating from a suitable donor a desired tissue sample of the biological material;
extracting the tissue with mildly alkaline hypotonic buffered solution of an endonuclease such as Benzonase(copyright) (a registered product of Merck KGaA, Darmstadt, Germany) and one or more non-denaturing detergents, preferably one or more anionic non-denaturing detergents;
optionally treating the tissue with a hypotonic buffered salt solution;
optionally treating the tissue with a hypertonic buffered salt solution;
washing the tissue with water, preferably ultrapure water followed by a water solution optionally containing one or more decontaminating agents including for example chlorine dioxide; and
storage in a sealed container in a storage solution optionally including isotonic saline, and/or one or more decontaminating agents.
The invention provides a process for preparing an devitalized soft tissue graft for implantation into a mammalian system and/or commercializable quantities of devitalized soft tissue grafts, including extracting a soft tissue sample with an extracting solution including one or more non-denaturing detergents, for example one or more non-denaturing anionic detergents, and one or more endonucleases, to produce extracted tissue; optionally treating the tissue with a hypertonic buffered salt solution to produce a treated tissue; washing the treated tissue with water followed by treating with a water solution of a decontaminating solution including one or more decontaminating agents to produce the devitalized soft tissue graft; and storing the devitalized soft tissue graft in a storage solution optionally comprising one or more decontaminating agents.
The invention provides a process for devitalizing soft tissue grafts without altering the matrix structure of the graft and without inhibiting subsequent recellularization of the soft tissue graft either in vitro, ex vivo, or in vivo.
The invention also provides a process for preparing an devitalized soft tissue graft for implantation into a mammalian system, including inducing a pressure mediated flow of an extracting solution including one or more non-denaturing detergents and one or more endonucleases, through soft tissue, to produce extracted tissue; inducing a pressure mediated flow of a decontaminating solution including one or more decontaminating agents through the treated tissue, to produce the devitalized soft tissue graft; and storing the devitalized soft tissue graft in a storage solution including one or more decontaminating agents.
The invention provides a process where the extracting solution is recirculated through the soft tissue graft.
The invention further provides a process where the treating solution is recirculated through the soft tissue graft.
The invention also provides a process where the decontaminating solution is recirculated through the soft tissue graft.
The invention provides a process for preparing an devitalized soft tissue graft for implantation into a mammalian system, including extracting a soft tissue sample with an extracting solution comprising one or more non-denaturing detergents and one or more endonucleases, to produce extracted tissue; washing said extracted tissue with a decontaminating solution comprising one or more decontaminating agents to produce said devitalized soft tissue graft; and storing said devitalized soft tissue graft in a storage solution wherein a devitalized soft tissue graft retaining large molecular weight proteins is produced.
The invention also provides a devitalization process does not employ a denaturing detergent.
The invention further provides a devitalization process which includes prior to said step of washing, first washing said extracted tissue with water.
The invention provides a process for preparing an devitalized soft tissue graft for implantation into a mammalian system, including first inducing a pressure mediated flow of an extracting solution including one or more non-denaturing detergents and one or more endonucleases, through soft tissue, to produce extracted tissue; inducing a pressure mediated flow of decontaminating solution optionally including one or more decontaminating agents, through said treated tissue; to produce said devitalized soft tissue graft; and storing said devitalized soft tissue graft in a storage solution.
The invention also provides a devitalization process where prior to the step of inducing, inducing a pressure mediated flow of a washing solution through said extracted tissue.
The invention also provides a devitalization process where extracting solution, and/or the decontaminating solution, and/or the washing solution, is recirculated through said soft tissue graft.
The invention provides a devitalization process where the non-denaturing detergent includes one or more detergents selected from the group consisting of: N-lauroylsarcosinate, a polyoxyethylene alcohol, a polyoxyethylene isoalcohol, a polyoxyethylene p-t-octyl phenol, a polyoxyethylene nonyphenol, a polyoxyethylene ester of a fatty acid, and a polyoxyethylene sorbitol ester.
The invention also provides a devitalization process where the decontaminating solution comprises ultrapure, endotoxin-free, water and/or water solutions of one or more decontaminating agents, where the decontaminating agents are non-reactive towards the one or more non-denaturing detergents.
The invention provides a devitalized tissue graft, including a soft tissue sample substantially free from reproductively viable and/or metabolically viable and/or cellular elements produced by the inventive process where recellularization of the devitalized tissue graft in vivo or in vitro, is enhanced.
The invention further provides a devitalized tissue graft, including a soft tissue sample substantially free from reproductively viable and/or metabolically viable and/or cellular elements.
The invention also provides a devitalized soft tissue sample which is a heart valve, and where the devitalized heart valve leaflets maintain normal coaptation.